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Scanning ion conductance microscopy: a convergent high-resolution technology for multi-parametric analysis of living cardiovascular cells.

Miragoli M, Moshkov A, Novak P, Shevchuk A, Nikolaev VO, El-Hamamsy I, Potter CM, Wright P, Kadir SH, Lyon AR, Mitchell JA, Chester AH, Klenerman D, Lab MJ, Korchev YE, Harding SE, Gorelik J - J R Soc Interface (2011)

Bottom Line: At the cellular level, heart failure leads to a pronounced loss of T-tubules in cardiac myocytes accompanied by a reduction in Z-groove ratio.The SICM pipette can be used for patch-clamp recordings of membrane potential and single channel currents.In conclusion, SICM provides a highly informative multimodal imaging platform for functional analysis of the mechanisms of cardiovascular diseases, which should facilitate identification of novel therapeutic strategies.

View Article: PubMed Central - PubMed

Affiliation: Cardiovascular Science, National Heart and Lung Institute, Imperial College London, , Dovehouse Street, London SW36LY, UK.

ABSTRACT
Cardiovascular diseases are complex pathologies that include alterations of various cell functions at the levels of intact tissue, single cells and subcellular signalling compartments. Conventional techniques to study these processes are extremely divergent and rely on a combination of individual methods, which usually provide spatially and temporally limited information on single parameters of interest. This review describes scanning ion conductance microscopy (SICM) as a novel versatile technique capable of simultaneously reporting various structural and functional parameters at nanometre resolution in living cardiovascular cells at the level of the whole tissue, single cells and at the subcellular level, to investigate the mechanisms of cardiovascular disease. SICM is a multimodal imaging technology that allows concurrent and dynamic analysis of membrane morphology and various functional parameters (cell volume, membrane potentials, cellular contraction, single ion-channel currents and some parameters of intracellular signalling) in intact living cardiovascular cells and tissues with nanometre resolution at different levels of organization (tissue, cellular and subcellular levels). Using this technique, we showed that at the tissue level, cell orientation in the inner and outer aortic arch distinguishes atheroprone and atheroprotected regions. At the cellular level, heart failure leads to a pronounced loss of T-tubules in cardiac myocytes accompanied by a reduction in Z-groove ratio. We also demonstrated the capability of SICM to measure the entire cell volume as an index of cellular hypertrophy. This method can be further combined with fluorescence to simultaneously measure cardiomyocyte contraction and intracellular calcium transients or to map subcellular localization of membrane receptors coupled to cyclic adenosine monophosphate production. The SICM pipette can be used for patch-clamp recordings of membrane potential and single channel currents. In conclusion, SICM provides a highly informative multimodal imaging platform for functional analysis of the mechanisms of cardiovascular diseases, which should facilitate identification of novel therapeutic strategies.

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Aorta cell alignment and architecture. (a) Intact hearts and attached thoracic aorta of 2-year-old landrace cross pig. (b) Representative SICM image of the inner part of the aorta where cells are organized in diffuse pattern. (c) SICM image of the outer part of the aorta shows a regularity of cell alignment, which indicates that this area is exposed to higher stress. Dashed arrows indicate blood flow direction. Effective pixel width in both images 625 nm, scan duration 13 min. Scanning pipette had a resistance of 100 MΩ and an estimated tip diameter of 100 nm. (A. Moshkov 2010, unpublished data.) (Online version in colour.)
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RSIF20100597F3: Aorta cell alignment and architecture. (a) Intact hearts and attached thoracic aorta of 2-year-old landrace cross pig. (b) Representative SICM image of the inner part of the aorta where cells are organized in diffuse pattern. (c) SICM image of the outer part of the aorta shows a regularity of cell alignment, which indicates that this area is exposed to higher stress. Dashed arrows indicate blood flow direction. Effective pixel width in both images 625 nm, scan duration 13 min. Scanning pipette had a resistance of 100 MΩ and an estimated tip diameter of 100 nm. (A. Moshkov 2010, unpublished data.) (Online version in colour.)

Mentions: Atherosclerosis is an even more important cause of death worldwide, with vessel inflammation, endothelial dysfunction and plaque formation in arterial walls. Plaques predominantly occur inside abrupt changes in vessels (branch points, bifurcations and the inner curvature of vessels) and the geometric specificity is probably owing to variation in shear stress as a function of flow velocity and viscosity [31,32] with plaques accumulating at atheroprone regions of low or oscillatory shear. Regions of high laminar shear stress are atheroprotected. The mechanism of atheroprotection by shear stress is yet to be fully determined. A difference in morphology has been identified using SICM. Owing to prominent undulations of the tissue surface, we narrowed the scan to an 80 × 80 µm region in SSCM mode [33]. Interestingly, using this approach we found differences in morphology between cells in the atheroprone (figure 3b, inner aortic arch) and the atheroprotected (figure 3c, outer aortic arch) regions of intact aorta. The inner arch cells were disordered with a cobblestone appearance, whereas the outer arch cells were elongated and aligned in the direction of blood flow, in accordance with findings using atomic force microscopy [34] and scanning electron microscopy [35].Figure 3.


Scanning ion conductance microscopy: a convergent high-resolution technology for multi-parametric analysis of living cardiovascular cells.

Miragoli M, Moshkov A, Novak P, Shevchuk A, Nikolaev VO, El-Hamamsy I, Potter CM, Wright P, Kadir SH, Lyon AR, Mitchell JA, Chester AH, Klenerman D, Lab MJ, Korchev YE, Harding SE, Gorelik J - J R Soc Interface (2011)

Aorta cell alignment and architecture. (a) Intact hearts and attached thoracic aorta of 2-year-old landrace cross pig. (b) Representative SICM image of the inner part of the aorta where cells are organized in diffuse pattern. (c) SICM image of the outer part of the aorta shows a regularity of cell alignment, which indicates that this area is exposed to higher stress. Dashed arrows indicate blood flow direction. Effective pixel width in both images 625 nm, scan duration 13 min. Scanning pipette had a resistance of 100 MΩ and an estimated tip diameter of 100 nm. (A. Moshkov 2010, unpublished data.) (Online version in colour.)
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3104336&req=5

RSIF20100597F3: Aorta cell alignment and architecture. (a) Intact hearts and attached thoracic aorta of 2-year-old landrace cross pig. (b) Representative SICM image of the inner part of the aorta where cells are organized in diffuse pattern. (c) SICM image of the outer part of the aorta shows a regularity of cell alignment, which indicates that this area is exposed to higher stress. Dashed arrows indicate blood flow direction. Effective pixel width in both images 625 nm, scan duration 13 min. Scanning pipette had a resistance of 100 MΩ and an estimated tip diameter of 100 nm. (A. Moshkov 2010, unpublished data.) (Online version in colour.)
Mentions: Atherosclerosis is an even more important cause of death worldwide, with vessel inflammation, endothelial dysfunction and plaque formation in arterial walls. Plaques predominantly occur inside abrupt changes in vessels (branch points, bifurcations and the inner curvature of vessels) and the geometric specificity is probably owing to variation in shear stress as a function of flow velocity and viscosity [31,32] with plaques accumulating at atheroprone regions of low or oscillatory shear. Regions of high laminar shear stress are atheroprotected. The mechanism of atheroprotection by shear stress is yet to be fully determined. A difference in morphology has been identified using SICM. Owing to prominent undulations of the tissue surface, we narrowed the scan to an 80 × 80 µm region in SSCM mode [33]. Interestingly, using this approach we found differences in morphology between cells in the atheroprone (figure 3b, inner aortic arch) and the atheroprotected (figure 3c, outer aortic arch) regions of intact aorta. The inner arch cells were disordered with a cobblestone appearance, whereas the outer arch cells were elongated and aligned in the direction of blood flow, in accordance with findings using atomic force microscopy [34] and scanning electron microscopy [35].Figure 3.

Bottom Line: At the cellular level, heart failure leads to a pronounced loss of T-tubules in cardiac myocytes accompanied by a reduction in Z-groove ratio.The SICM pipette can be used for patch-clamp recordings of membrane potential and single channel currents.In conclusion, SICM provides a highly informative multimodal imaging platform for functional analysis of the mechanisms of cardiovascular diseases, which should facilitate identification of novel therapeutic strategies.

View Article: PubMed Central - PubMed

Affiliation: Cardiovascular Science, National Heart and Lung Institute, Imperial College London, , Dovehouse Street, London SW36LY, UK.

ABSTRACT
Cardiovascular diseases are complex pathologies that include alterations of various cell functions at the levels of intact tissue, single cells and subcellular signalling compartments. Conventional techniques to study these processes are extremely divergent and rely on a combination of individual methods, which usually provide spatially and temporally limited information on single parameters of interest. This review describes scanning ion conductance microscopy (SICM) as a novel versatile technique capable of simultaneously reporting various structural and functional parameters at nanometre resolution in living cardiovascular cells at the level of the whole tissue, single cells and at the subcellular level, to investigate the mechanisms of cardiovascular disease. SICM is a multimodal imaging technology that allows concurrent and dynamic analysis of membrane morphology and various functional parameters (cell volume, membrane potentials, cellular contraction, single ion-channel currents and some parameters of intracellular signalling) in intact living cardiovascular cells and tissues with nanometre resolution at different levels of organization (tissue, cellular and subcellular levels). Using this technique, we showed that at the tissue level, cell orientation in the inner and outer aortic arch distinguishes atheroprone and atheroprotected regions. At the cellular level, heart failure leads to a pronounced loss of T-tubules in cardiac myocytes accompanied by a reduction in Z-groove ratio. We also demonstrated the capability of SICM to measure the entire cell volume as an index of cellular hypertrophy. This method can be further combined with fluorescence to simultaneously measure cardiomyocyte contraction and intracellular calcium transients or to map subcellular localization of membrane receptors coupled to cyclic adenosine monophosphate production. The SICM pipette can be used for patch-clamp recordings of membrane potential and single channel currents. In conclusion, SICM provides a highly informative multimodal imaging platform for functional analysis of the mechanisms of cardiovascular diseases, which should facilitate identification of novel therapeutic strategies.

Show MeSH
Related in: MedlinePlus